Chromatography Forum

Hello everyone,

Currently i'm dealing with the dissolution of amlodipine besylate and benazepril hydrochloride capsules,

the mobile phase used is normal TEA buffer with a pH of 3( imean just TEA in Milli-q water and then the pH is adjusted to 3 with OPA)

the column used is 100 x 4.6 mm, 3um hypersil BDS c18

usually the Benazepril peak is eluted at an RT of 6 mins and where as Amlo is eluted at around 8.5 mins, there's no shift in the RT for the first 5 injections of the standard

but when the samples are being injected the amlo peak gradually moves towards the benazepril peak and due to this the brackettings are failing

what could be the reason?

if you say column stabilization .... the RT shift is observed even after the column is stabilised for 2 hours


if you say buffer strength( i mean due to poor buffer strength)... well its a validated method

i'm not understanding where the problem lies

it could be termed as an analytical error if it happens once, but the same problem is seen even after changing the analyst and even the chromatographic system


plz respond immediately

Since you have run this several time, the retention of the amlodipine peak must increase to its original value. What is required (new column, new batch of mobile phase, wait a day, . . .) to accomplish this?

What is the organic solvent used in the mobile phase, and what is its concentration?

It can take well over 2 hours for TEA to fully finish altering the alpha of a column. Try making a small batch of MP w/ 2 or 3x your desired TEA concentration and recirculating it through the column at a modest flow rate overnight. Whatever your retention times are after switching back to regular MP should be stable after having passed 5-10 column volumes (if it's a TEA/column issue).

Since you have run this several time, the retention of the amlodipine peak must increase to its original value. What is required (new column, new batch of mobile phase, wait a day, . . .) to accomplish this?

i've continuously injected the standards for 24 hrs, the RT shift is first seen from 8.5 to 7.1min then fron there to 9.6 mins

then i've changed the buffer ... imean i've run it using a KH2PO4 buffer of pH 3( 2.72 gms of KH2PO4+ 2 mL TEA in 1000mL of Milli-q water and the pH adjusted to 3 using OPA)


the RT shift is Observed but its around 0.2 to 0.3 mins from the initial

but i just wanted to know where lies the problem in the earlier method i've tried it on a new hypersil column, even then the RT shift was seen!!!

i mean just curious coz i was told that its a validated method

I think you've answered your own question: it was the buffer. (Bad methods get validated all too often! )

Right. Adding acid to adjust a pH in this case can leave you with considerable variations in ionic strength besides having a variable buffer concentration (phosphate in both cases). Also, the second phosphate buffer probably has a much higher concentration, a higher ion concentration seems to let columns come to equilibrium faster (probably via the obliteration of silanol ion effects?).